Significances of red cell bound immunoglobulin G as detected by flow cytometry in patients with Coombs-negative immune hemolysis.

OBJECTIVES: This study was to investigate the use of flow cytometry for detection and quantitation of red blood cells (RBC) bound IgG in immune hemolysis of patients with autoimmune hemolytic anaemia (AIHA) and systematic lupus erythematosus (SLE). BACKGROUND: Two to ten percent of patients with warm-autoimmune hemolytic anaemia (WAIHA) exhibit a negative direct Coombs test. Flow cytometry has been applied to detect RBC bound IgG with high accuracy, reproducibility and sensitivity. METHODS: In this study 45 and 75 patients with AIHA and SLE, respectively were evaluated for RBC bound IgG by direct Coombs test and flow cytometry. RESULTS: Seventy-one percent (32/45) and 31% (23/75) of patients with AIHA and SLE respectively, had laboratory evidence of hemolysis. A positive flow cytometry, as defined by mean fluorescent intensity (MFI) values >0·21 and IgG molecules >28, was found in 4 of 32 (12·5%) and 4 of 23 (17·4%) patients with AIHA and SLE who had hemolysis with a negative direct Coombs test. There were very strong and strong correlations between the strength of direct Coombs test with MFI values and IgG molecules in patients with AIHA and SLE, respectively. CONCLUSION: Flow cytometry can be applied in the diagnosis of Coombs-negative hemolytic anaemia in patients with AIHA and SLE.

Emergency splenectomy in adult idiopathic thrombocytopenic purpura. A report of seven cases.

Seven adult patients with idiopathic thrombocytopenic purpura underwent emergency splenectomy. Six were female and one was a male, aged 16 to 61 years. All of them had a life-threatening episode. Six patients had progressive intracranial bleeding and one had postsurgical intra-abdominal bleeding. All patients were saved by surgery, except one for whom operation was delayed. There was no postoperative bleeding or surgical complication. Immediate splenectomy should be the treatment of choice in any patient with idiopathic thrombocytopenic purpura complicated by life-threatening hemorrhage.

Serum levels of tumor necrosis factor-alpha, interleukin-1, and interferon-gamma in beta(o)-thalassemia/HbE and their clinical significance.

Serum levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1alpha (IL-1alpha), and interferon-gamma (IFN-gamma) were estimated by conventional ELISA kits in 60, 42, and 58 Thai patients, respectively, with beta(o)-thalassemia HbE and found to be above the normal range in 13%, 21%, and 33% of the patients, respectively. Using high-sensitivity ELISA systems, an additional 10 beta(o)-thal/HbE patients were compared with 9 controls for concentrations of circulating TNF-alpha and IL-1beta, and 9 and 5 patients, respectively, but only 1 and none of the controls, respectively, showed values above the normal ranges. In patients with abnormally high IFN-gamma levels, basal hemoglobin values were significantly lower than in those with normal levels of the cytokine (mean +/- SEM: 6.03+/-0.24 vs. 7.08+/-0.18, p < 0.05), although circulating concentrations of soluble transferrin receptors (sTrF) and absolute reticulocyte counts were similar in the two groups. Patients with raised or normal levels of TNF-alpha, IL-1alpha, or IL-1beta had similar basal hemoglobin values. In a phagocytosis assay, monocytes of patients with raised serum levels of IFN-gamma showed significantly more attached or ingested IgG-coated red cells than those of patients with normal concentrations of the cytokine (mean +/- SEM: 192+/-22 vs. 140+/-14 per 100 monocytes, p < 0.05). Moreover, in 3 of 4 of the former patients, the number of attached or ingested IgG-coated red cells per 100 monocytes was above the 95% reference limit for the latter patients. The results suggest that IFN-gamma aggravates the anemia of beta(o)-thal/HbE by activating mononuclear phagocytes for destruction of red cells but not by inhibiting erythropoiesis. The elevated serum levels of TNF-alpha and IL-1 could contribute to complications of the disease, such as cachexia and thromboembolic phenomena.

Cloning of the glucose 6-phosphate dehydrogenase gene from Plasmodium falciparum.

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is one of the human genetic traits that confer relative resistance against malaria caused by Plasmodium falciparum. It has been previously shown that this organism, during its intraerythrocytic development, produces its own G6PD, which has properties different from those of human G6PD. In order to investigate the role of this enzyme in parasite-host cell interactions, we have isolated the G6PD gene from Plasmodium falciparum as a set of overlapping lambda gt11 clones. By sequence analysis we have found a single open reading frame, uninterrupted by introns, coding for a protein of 910 amino acids, almost twice as long as any previously sequenced G6PD molecule. The P. falciparum G6PD mRNA is 5.1 kb in size and has an exceptionally long 5' untranslated region of some 1000 nucleotides. We have mapped the G6PD gene to chromosome 14. The C-terminal portion of the predicted protein, from amino acid 310-910 (except for an 'insert' of 62 amino acids), has 39% homology to human G6PD, with a number of characteristic, fully conserved peptides. The N-terminal portion of the predicted protein has no homology to G6PD, but it contains a peptide in which 7 out of 12 amino acids are identical to the putative glutathione binding site of human glutathione S-transferase.

Analysis of the alloreactive T cell repertoire in man. I. Differences in precursor frequency for cytotoxic T cell responses against allogeneic MHC molecules in unrelated individuals.

We demonstrate here that in man the frequency of cytotoxic T cells specific for a given set of allo-major histocompatibility complex (MHC) antigens varies among unrelated individuals. This has been established by limiting dilution analysis of human peripheral blood lymphocytes (HPBL) in the presence of irradiated autologous filler cells, T cell growth factors, and irradiated HPBL carrying allo-MHC antigens. Two unrelated individuals were tested against the same panel of allo-MHC antigens. We have been able to identify frequencies of T cells ranging from 1:5,000 to 1:20,000(high), 1:20,000 to 1:60,000(intermediate) and 1:60,000 to 1:100,000(low). In some cases, the precursor frequency of cytotoxic T cells was so low that it was considered to represent nonresponsiveness. The clear differences in precursor frequency suggest that this system is suitable for further analysis of the allo-MHC-specific repertoire in man.

Clinical paroxysmal nocturnal hemoglobinuria is the result of expansion of glycosyl-phosphatidyl-inositol-anchored protein-deficient clone in the condition of deficient hematopoiesis.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired, clonal hematopoietic stem cell disorder in which PIG-A, gene essential for the biosynthesis of the glycosyl-phosphatidyl-inositol (GPI) anchor, is somatically mutated. Absence of GPI-linked proteins from the surface of blood cells is characteristic of the PIG-A mutant (PNH) clone and is also accountable fo certain manifestations, such as intravascular hemolysis. It is unclear how the PNH clone expands and comes to dominate hematopoiesis. In this study, CD34+ cells--committed progenitors (colony-forming cells) representing immature hematopoietic stem cells--and reticulocytes representing the differentiated erythroid cells were quantitated in peripheral blood of patients with PNH. Compared with normal controls (n = 29), CD34+ cell levels were significantly lower in PNH patients who did not have preexisting aplastic anemia (AA) (n = 12) (2.47+/-1.23 versus 4.68+/-1.05 x 106/L, mean +/- standard error; P = .022). PNH patients with precedent aplastic anemia (AA+/PNH) showed markedly low CD34+ cell levels compared with normal control subjects (0.6+/-0.29 versus 4.68+/-1.05 x 10(6)/L; P = .0001). In addition, colony-forming cells from PNH patients were significantly decreased compared with those from normal volunteers (erythroid burst-forming units, 2.8+/-1.2 versu 25.6+/-6.2/5 x 10(5) mononuclear cells; P = .0006; and granulocyte/macrophage colony-forming units, 1.2+/-0.5 versus 13.3+/-3.0/ 5 x 10(5) mononuclear cells; P = .0006). These findings occur in both aplastic and hemolytic types of PNH, suggesting hematopoietic failure in PNH. On the contrary, the numbers of reticulocytes and the reticulocyte production index of PNH patients were significantly higher than those of normal persons and comparable to those from patients with autoimmune hemolytic anemia, indicating accelerating erythropoiesis in PNH. The degree of reticulocytosis correlated well with the proportion of CD59- (PNH) reticulocytes. All of the findings suggest that in the condition of deficient hematopoiesis, the PNH clone arising from the mutated hematopoietic stem cell expands and maintains a substantial proportion of the patient's hematopoiesis.

Fatal arteritis due to Pythium insidiosum infection in patients with thalassaemia.

Six thalassaemic patients had a distinct clinical syndrome characterized by progressive ischemia of the lower extremities, with ascending arteritis and thrombosis of the main arteries of the lower limbs. With periodic acid Schiff and Gomori's methenamine silver staining a large number of hyphae were revealed in the arterial wall and the outer part of the thrombus. Pythium insidiosum was isolated from 3 patients. The clinical course of the disease was progressive gangrene of the extremities and the patients invariably died when the infectious process reached the bifurcation of the aorta. There is no effective antimicrobial agent for the syndrome and radical amputation was the only method to ensure survival of the patients. P. insidiosum infection should be considered in thalassaemic patients with leg ulcers or arterial occlusion of the lower limbs.

Therapy of chronic idiopathic thrombocytopenic purpura in adults: experiences from Thailand.

Results of different types of treatment in 416 adults with chronic idiopathic thrombocytopenic purpura (ITP) were analyzed. Of 368 patients treated with corticosteroids, 278 (77%) achieved complete response (CR ie, normalization of platelet count). However, only 18% of patients had continued complete response (CCR). Results of splenectomy was analyzed in 126 patients, 49% of them achieved CCR and 76% of them had beneficial effects from the operation. Emergency splenectomy was a life saving procedure in 8 of 11 patients with life-threatening hemorrhage. Vinca alkaloids gave a 50% transient CR. Immunosuppressive agents (cyclophosphamide, azathioprine) resulted in only 11% CCR.

High-dose intravenous immunoglobulin in the management of immune hemolysis in patients with thalassemic disease: factors which determine refractoriness.

We report our experience with high dose intravenous immunoglobulin (IVIg) in 3 thalassemic patients who had evidence of possible immune hemolysis. In 2 patients who had serious sepsis, their responses to IVIg were only partial and transient. The other patient who had marked splenomegaly had no evidence of response to IVIg. Both serious infections and large spleen may hamper the effect of IVIg and should be considered before IVIg is to be used in thalassemia.

Erythrocyte glucose-6-phosphate dehydrogenase and pyruvate kinase activities in hemoglobin H disease.

Erythrocyte glucose-6-phosphate dehydrogenase (G6PD) and pyruvate kinase (PK) activities were studied in hemoglobin H (HbH) patients by spectrophotometric method, cytochemical method and the methemoglobin reduction (MR) test for the detection of heterozygous G6PD deficiency. G6PD deficiency was found in 7 of 64 cases (10.9%), including 3 cases of genotype alpha 1/alpha 2 and 4 cases of genotype alpha 1/CS. None of the HbH patients was found to be PK-deficient. Spectrophotometrically determined G6PD and PK activities were significantly higher in HbH patients than in normals (p less than 0.001), whereas the MR test yielded a significantly lower percentage of residual methemoglobin in HbH patients than in normals (p less than 0.05). All three methods were efficient in the detection of hemizygous G6PD deficiency in HbH patients, but not in G6PD-deficient females.

PIG-A gene abnormalities in Thai patients with paroxysmal nocturnal hemoglobinuria.

Deficient biosynthesis of the glycosyl phosphatidyl inositol (GPI)-anchor in blood cells is implicated in the pathogenesis of paroxysmal nocturnal hemoglobinuria (PNH). Abnormal clonal cells appear in various hematopoietic cell lineages, suggesting that PNH arises as a result of somatic mutation occurred at the multipotential hematopoietic stem cell stage. We previously cloned a gene which is responsible for PNH. The gene termed PIG-A (for Phosphatidyl Inositol Glycan-class A) participates in the early step of GPI-anchor biosynthesis. Studies with cell lines and granulocytes from patients with PNH revealed that in all cases so far characterized, PIG-A is the target for the somatic mutation. In the present study, we analyzed PIG-A abnormality in granulocytes from 14 Thai-patients with PNH. PIG-A RNA was reversed transcribed and the coding region was amplified by polymerase chain reaction and cloned into plasmids. The cDNA thus obtained and genomic DNA were analyzed by mutation detection enhancement gel electrophoresis and sequencing. The assessment of function of PIG-A cDNA was based on the ability to correct the phenotype of a PIG-A deficient cell line after transfection. The result showed that all patients had PIG-A abnormality. Three patients had size abnormality of PIG-A transcripts caused by mutations at the splicing sites in the genomic DNA level. Eleven patients had PIG-A transcripts of normal sizes but had mutations in the coding region which included small deletions and insertions. Taken together with the result from Japanese and British patients, the PIG-A somatic mutations in patients with PNH are small mutations widely distributed throughout coding region and the splicing sites.

Genotypic and phenotypic implications in paroxysmal nocturnal hemoglobinuria (PNH): a preliminary investigation.

The genetic and biochemical defects underlying paroxysmal nocturnal hemoglobinuria (PNH) have recently been elucidated. The deficiency of the surface expression of glycosylphosphatidylinositol (GPI)-anchored proteins caused by a somatic mutation of the PIG-A gene, an X-chromosomal gene that participates in the first step of the GPI anchor synthesis, has been shown to be responsible for PNH in all patients. The mutations of PIG-A studied to date are highly heterogeneous. They are however mainly of the frameshift type (61.5%). The characteristic abnormalities of PNH phenotypes has also been shown especially by DAF- and/or CD59-based fluorescent immunocytometry. A great degree of heterogeneity in the patterns and levels of expression of GPI-anchored proteins in various cell types was demonstrated indicating a discrepancy of lineage involvement. In this investigation, major blood cell populations, i.e erythrocytes and granulocytes were analyzed immunophenotypically, the mutations of PIG-A were identified by heteroduplex analysis and nucleotide sequencing and the consequences of PIG-A mutations were observed. All the mutations identified in 9 patients with PNH resulted in complete loss of function as clones of affected granulocytes completely negative for CD59 expression were shown in all patients. Interestingly, granulocytes in these patients contained variable proportions of affected cells varied from 50% to 100% and four of the patients had erythrocytes with diminished expression of GPI-anchored DAF and CD59 coexisting with normal and completely negative cells. Immunophenotypic analysis of reticulocytes in peripheral blood of patients with PNH demonstrated the conserved patterns of DAF and CD59 expression in circulating erythroid cells and the discrepancies between granulocytic and erythroid lineages. These findings suggested that the characteristics of abnormal phenotypes which appear to be highly variable between different hematopoietic lineages are not solely caused by mutation of PIG-A but are influenced by other factor(s).

Cross over placebo control trial of dilazep in beta-thalassemia/hemoglobin E patients.

An attempt was made to find better symptomatic treatment for beta-thalassemia/hemoglobin E (beta-thal/Hb E) patients in order to reduce their blood demand. Oral administration of dilazep was prescribed for these patients and a clinical trial was conducted over a 2-year period as a cross over placebo control study. Seventeen beta-thal/Hb E patients were enrolled in the study. All of them received dilazep and placebo for 10 months at different periods of time and were taken care of by the same doctor throughout the study. The blood demand of the same patients during the period of receiving dilazep with the period of receiving placebo, was 1.5 +/- 1.8 U/10 months versus 2.2 +/- 2.6 U/10 months, respectively. Thus dilazep showed a benefit in decreasing the blood demand by about 50% although the results did not reach statistical significance (p = 0.1). There was a statistical difference in hemoglobin concentration of the patients receiving dilazep compared with placebo (p = 0.038). While receiving dilazep the mean +/- SD hemoglobin level was 5.82 +/- 0.8 g/dl, significantly higher than while receiving placebo (5.66 +/- 0.9 g/dl) (p = 0.038). The liver, and renal function tests, and cardiac enzyme levels of the patients showed no significant changes throughout the study. However, one case had a problem with bleeding following tooth extraction whilst receiving dilazep and needed 1 unit of blood transfusion. In conclusion, administration of dilazep to patients with beta-thal/Hb E increased the patients' hemoglobin and reduced their blood demand with few side effects.

A double-blind placebo control trial of dilazep in beta-thalassemia/hemoglobin E patients.

Since the obtained results from the pilot study indicated that dilazep which was a membrane stabilizer would be benefit to treatment and prevention of anemia and chronic leg ulcer in beta-thalassemia/hemoglobin E (beta-thal/HbE) patients, the authors had continued the study in a second phase, ie a double blind placebo control trial. Twenty-seven beta-thal/HbE patients were recruited in the study. Eight patients who suffered from chronic leg ulcer were given dilazep. The rest of patients were given dilazep or placebo according to a randomized table. Hence, 16 patients received dilazep and 11 received placebo. When we compared the number of unit of blood transfusion, hemoglobin level, 2-3 DPG and P50 value between the dilazep and placebo groups using unpaired t-test, we found that there were no statistical differences in any of the parameters. However, when we compared the data within the group using paired t-test, there was statistical decrease in blood requirement after treatment in the dilazep group (p < 0.05). Concerning with the treatment of chronic leg ulcer, 3 in 8 patients were completely healed within 3 months, 4 in 8 patients were improved and 1 in 8 patients was not improved. There were complaints of skin itching and mild epigastric pain in placebo group but the liver function tests, kidney function tests and cardiac enzyme did not significantly change during the medication.

Severe hyperbilirubinemia in glucose-6-phosphate dehydrogenase deficient patients during viral hepatitis.

Nine G-6-PD subjects developed acute hemolysis and severe hyperbilirubinemia (up to 61.1 mg/dl) following viral hepatitis. All except one had fever at presentation. Neutrophilic leukocytosis was a common feature. Elevation of both alanine aminotransferase (SGPT) and extremely high level of aspartate aminotransferase (SGOT) were prominent. Three developed acute renal failure. All patients survived, one after peritoneal dialysis. Recognition of the clinical picture is essential to prevent serious complications and for successful management.

Outcomes of pregnancy in adult idiopathic thrombocytopenic purpura.

The outcomes of 39 pregnancies in 36 idiopathic thrombocytopenic purpura (ITP) patients were analysed. Among the 36 pregnant patients, 23 had active ITP (platelet count < 100,000 per mm3), 12 had inactive ITP, during pregnancies, while one patient had active and inactive ITP during each of her two pregnancies. Maternal bleeding during delivery occurred more commonly in thrombocytopenic patients and was more frequent by cesarean section than by vaginal delivery. There was a 56 per cent incidence of neonatal thrombocytopenia. Maternal platelet count had no predictive value for neonatal thrombocytopenia. There was no serious bleeding among neonates born from thrombocytopenic mothers. In our experience, vaginal delivery was a relatively safe procedure for delivering small neonates from multiparous ITP mothers. Cesarean section should be used to deliver relatively big neonates especially from primigravida ITP mothers and splenectomy should be done at the same time.

Pulmonary dirofilariasis in a patient with multisystem Langerhans cell histiocytosis--the first reported case in Thailand.

Despite a high prevalence of canine dirofilariasis, there is no case of pulmonary dirofilariasis reported from Thailand. We herein report a case of multisystem Langerhans cell histiocytosis who had an incidental pulmonary dirofilariasis found at the time of autopsy as a solitary nodule at the periphery of the right lower lobe. This is the first reported case in Thailand. Association between pulmonary dirofilariasis and Langerhans cell histiocytosis has not been described before in the literature.